Ascorbic acid production from D-glucose in yeast

Abstract

Herein is disclosed a method of generating ascorbic acid from yeast transformed with a mannose epimerase. In a further embodiment, the yeast can be further transformed with a myoinositol phosphatase. In the method, the transformed yeast can produce L -ascorbic acid from D -glucose. The transformed yeast has been observed to have increased growth rate, cell density, or survival when cultured on appropriate media.

1 . A method of generating  L -ascorbic acid, comprising: 
 a) obtaining a recombinant yeast functionally transformed with a coding region encoding a mannose epimerase (ME);    b) culturing the recombinant yeast in a medium comprising  D -glucose, thereby forming  L -ascorbic acid, and    c) isolating the  L -ascorbic acid.    
     
     
         2 . The method of  claim 1 , wherein the recombinant yeast is further functionally transformed with a coding region encoding a myoinositol phosphatase (MIP).  
     
     
         3 . The method of  claim 1 , wherein the yeast belongs to the genus  Saccharomyces, Zygosaccharomyces, Candida, Hansenula, Kluyveromyces, Debaromyces, Nadsonia, Lipomyces, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Cryptococcus, Trichosporon, Aureobasidium, Lipomyces, Phaffia, Rhodotorula, Yarrowia,  or  Schwanniomyces.    
     
     
         4 . The method of  claim 3 , wherein the yeast belongs to the species  S. cerevisiae, K. lactis,  or  Z. bailii.    
     
     
         5 . The method of  claim 4 , wherein the yeast is selected from  S. cerevisiae  strain GRF18U;  S. cerevisiae  strain W3031B, BY4742, and YML007w,  K. lactis  strain CBS2359, or  Z. bailii  strain ATCC 60483.  
     
     
         6 . The method of  claim 1 , wherein the ME has at least about 95% identity with SEQ ID NO:1.  
     
     
         7 . The method of  claim 6 , wherein the ME has at least about 98% identity with SEQ ID NO:1.  
     
     
         8 . The method of  claim 2 , wherein the MIP has at least about 95% identity with SEQ ID NO:2.  
     
     
         9 . The method of  claim 8 , wherein the MIP has at least about 98% identity with SEQ ID NO:2.  
     
     
         10 . The method of  claim 1 , wherein the yeast is further functionally transformed with a coding region encoding an enzyme selected from  L -galactose dehydrogenase (LGDH),  L -galactono-1,4-lactone dehydrogenase (AGD),  D -arabinose dehydrogenase (ARA),  D -arabinono-1,4-lactone oxidase (ALO), or  L -gulono-1,4-lactone oxidase (GLO).  
     
     
         11 . The method of  claim 1 , wherein the coding region is linked to a promoter active in the yeast.  
     
     
         12 . The method of  claim 11 , wherein the promoter is the  S. cerevisiae  triosephosphateisomerase (TPI) promoter.  
     
     
         13 . The method of  claim 1 , wherein the isolating step comprises lysing the yeast.  
     
     
         14 . The method of  claim 13 , wherein the isolating step further comprises centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, liquid-liquid extraction, crystallization, enzymatic treatment with nuclease or protease, or chromatography.  
     
     
         15 . The method of  claim 1 , wherein the isolating step comprises chromatography, activated carbon, microfiltration, ultrafiltration, nanofiltration, liquid-liquid extraction, or crystallization.  
     
     
         16 . A recombinant yeast, wherein the yeast is functionally transformed with a coding region encoding a mannose epimerase (ME).  
     
     
         17 . The recombinant yeast of  claim 16 , wherein the recombinant yeast is further functionally transformed with a coding region encoding a myoinositol phosphatase (MIP).  
     
     
         18 . The recombinant yeast of  claim 16 , wherein the ME has at least about 95% identity with SEQ ID NO:1.  
     
     
         19 . The recombinant yeast of  claim 17 , wherein the MIP has at least about 95% identity with SEQ ID NO:2.  
     
     
         20 . The recombinant yeast of  claim 16 , wherein the yeast is further functionally transformed with a coding region encoding an enzyme selected from  L -galactose dehydrogenase (LGDH),  L -galactono-1,4-lactone dehydrogenase (AGD),  D -arabinose dehydrogenase (ARA),  D -arabinono-1,4-lactone oxidase (ALO), or  L -gulono-1,4-lactone oxidase (GLO).  
     
     
         21 . A method of increasing the production, productivity, or yield of a product produced by a microorganism during fermentation, comprising: 
 functionally transforming the microorganism with a coding region encoding a mannose epimerase (ME).    
     
     
         22 . The method of  claim 21 , further comprising functionally transforming the microorganism with a coding region encoding a myoinositol phosphatase (MIP).  
     
     
         23 . The method of  claim 21 , wherein the microorganism is selected from the group consisting of bacteria, yeast, filamentous fungi, animal cells, and plant cells.  
     
     
         24 . The method of  claim 23 , wherein the yeast belongs to the species  S. cerevisiae, K. lactis,  or  Z. bailii.    
     
     
         25 . The method of  claim 24 , wherein the yeast belongs to the strain  S. cerevisiae  GRF.